Supramolecular complexation of phenylephrine by cucurbit[7]uril in aqueous solution

Cucurbiturils (CBn) are known to establish stable host–guest complexes with a variety of drug molecules. Herein, the supramolecular complexation between cucurbit[7]uril (CB7) and phenylephrine hydrochloride is reported in aqueous solution. Phenylephrine forms inclusion complex with CB7 with high binding affinity (Kaffinity = 4.0 × 106 M−1), which allows for the development of a fluorescence-based sensing assay applying the dye displacement strategy. The structure of the host–guest inclusion complex is investigated by 1H NMR spectroscopy, in which complexation-induced chemical shifts indicate the immersion of the aromatic ring inside the hydrophobic cavity of CB7. Density functional theory (DFT) calculations support the 1H NMR results, and reveal that the complex is stabilized through intermolecular interactions between the polar groups on the phenylephrine and the carbonyl rims of CB7, as well as the hydrophobic effect. Moreover, preferential binding of phenylephrine in its protonated over the neutral form results in a complexation-induced pKa shift.


Introduction
Cucurbit[n]urils, CBn, are a family of interesting macrocyclic host molecules that are water-soluble. 1,2CBn and other homologues are synthesized by the condensation of glycoluril and formaldehyde in strongly acidic media, 3,4 where the reaction composition representing the molecules was rst identied by crystallography. 5CBn are composed of n (5-8, 10 and 14) repeating glycoluril units linked via two methylene bridges on each side, which results in a barrel-shaped molecules with two identical carbonyl rims and lipophilic cavity with low polarity and polarizability. 1CBn have found great interest due to their ability to encapsulate a wide spectrum of guest molecules within the hydrophobic cavity with high association constants. 1,2The host-guest complexation of CBn is driven by the hydrophobic effect and non-covalent interactions. 62][13][14][15] Several examples on the complexation of drug molecules with different CBn hosts in aqueous solution are documented in the literature. 16][19] In this work, we study the molecular recognition of phenylephrine by CB7 through host-guest complexation (Fig. 1).Indeed, CB7 was selected due to its moderate cavity size (242 Å) that ts small organic molecules, as well as its relatively high water solubility. 20Phenylephrine is used effectively to treat congestion, vasomotor rhinitis, hemorrhoids and hypotension, and commonly prescribed an alternative to pseudoephedrine. 21,22The supramolecular complexation is investigated by using different spectroscopic techniques and the structure of the host-guest complex is elucidated by nuclear magnetic resonance (NMR) spectroscopy and veried by quantum-chemical calculations.
of important pharmacological molecules that act as neurotransmitters and hormones. 264][35] Phenylephrine showed an absorption maximum in UV-range (l max = 275 nm, in acidic solution and l max = 290 nm, in basic solution).The addition of CB7 to a phenylephrine solution resulted in a hyperchromic shi, which in part a consequence of the CB7 absorption in the same region.Therefore, indicator displacement assay was chosen to determine the binding affinity of phenylephrine to CB7. 33,36 Berberine chloride (Fig. 1) was selected as a sensitive dye, which is known to form stable inclusion complex with CB7. 37In aqueous solution, berberine showed a weak uorescence emission (l em = 495 nm), which is signicantly enhanced upon complexation with CB7, due to the relocation into the hydrophobic cavity and the rigid connement. 378][39] The preformed CB7$berberine complex can potentially be used as a reporter pair for the sensing of optically transparent guest molecules. 34The addition of a competitive binder (e.g., phenylephrine) results in the displacement of the dye (i.e., berberine) and the restoration of the original uorescence emission.Fig. 2 illustrates the principle of uorescence-based dye displacement.
The uorescence displacement titration of phenylephrine is shown in Fig. 3A.The uorescence intensity of complexed berberine decreased upon the addition of phenylephrine.This indicates the displacement of berberine by phenylephrine.The emission intensity was then monitored as a function of the phenylephrine concentration and tted according to a 1 : 1 binding model, which resulted in a binding constant of 4.0 × 10 6 M −1 (Fig. 3B).The binding affinity of phenylephrine to CB7 is found to be higher than that of dopamine 40 (a structurally similar catecholamine), which might be attributed to the position of a hydroxyl group at the side chain of phenylephrine rather than at the phenyl ring in dopamine.
The sensitive uorescence response of CB7$berberine to the phenylephrine could potentially be used for sensing purposes in aqueous solutions.A linear uorescence response was observed in the concentration range of 0 to 30 mM of phenylephrine (Fig. 4, R 2 = 0.997).Accordingly, the limits of detection (LOD) and quantication (LOQ) were estimated as 0.58 and 1.77 mg L −1 .This represents a new uorescence-based sensing method for phenylephrine.
CBn are known as strong binders for a variety of neutral and cationic guest molecules in aqueous solution. 2The preferential binding of the cationic form of guest molecules (e.g., ammonium-based) over their neutral form, modulates their acid dissociation constants (pK a ), leading to a complexationinduced pK a shis. 9,15,41,42The pK a values of free and complexed phenylephrine were determined by monitoring the absorbance as a function of pH (Fig. 5).The UV-spectra of free phenylephrine showed an absorption band with l max of 275 nm at low pH (<7), which gradually shis to a longer wavelength (bathochromic shi) as a function of increasing the pH (l max of 290 nm), with two isosbestic points at 259 and 278 nm.The pK a of phenylephrine was determined as 9.35.Similarly, the pK a of complexed phenylephrine was measured in the presence of 1.0 mM CB7 to ensure a high degree of complexation at elevated pH values.No signicant change in the l max of phenylephrine was observed in the presence of CB7 in acidic solution, while a slight bathochromic shi (5 nm) was obtained in basic solution.CB7-complexed phenylephrine showed an increase in the pK a value to 10.52 (DpK a of 1.17).This shi to a higher pK a value indicated an increased basicity of phenylephrine in the presence of CB7 as consequence of the stabilization of the protonated form due to the ion-dipole interactions between the   ammonium group in the phenylephrine and the carbonyl rim of CB7 (see below).
The formation of host-guest complexes between phenylephrine and CB7, in acidic aqueous solution, was investigated using 1 H NMR spectroscopy, which is a powerful technique used to extract structural information.The formation of inclusion complexes in aqueous solution is usually associated with complexation-induced chemical shis. 43In detail, we rely on chemical shi values that indicate whether the guest molecule or part of it is located inside the hydrophobic cavity or closer to the portals of the macrocyclic host.This is usually obtained by measuring the difference in 1 H NMR chemical shis of the bound (d bound ) and free guest (d free ), which can be expressed as follows Dd = d bound − d free .In the case of CBn, the host protons are less informative, due to the absence of protons inside the cavity.Changes in the chemical shi of the guest protons can give a clear evidence for complexation.When, Dd > 0, this means that the protons of the guest are located near to the carbonyl groups of CB7 and appear downeld shied.When Dd < 0, the protons of the guest are positioned inside the lipophilic cavity of CB7 and appear upeld shied.Fig. 6 displays the 1 H NMR spectra of free phenylephrine, free CB7, and their complex, dissolved in D 2 O.The phenylephrine signals were assigned as shown in the chemical structure Fig. 6B.The formation of the inclusion complex between CB7 and phenylephrine affects the proton chemical shis based on their location within the cavity of CB7.The relocation of the guest into the hydrophobic cavity of CB7 induced signicant upeld shis due to the shielding effect.In contrast, protons that are located at the carbonyl rims are usually downeld shied compared to the uncomplexed ones.The aromatic protons 4-7 were upeld shied upon complexation (Dd = −0.6 to −1 ppm), which indicated that the phenyl ring is positioned inside the cavity of CB7.Similarly, proton 3 experienced an upeld shi.In contrast, the methyl protons (1) were downeld shied as expected from their location near the CB7 portal.The methylene protons (2) split upon complexation, which revealed different environments within the complex.These data revealed that the protonated ammonium group will be docked at the portal to enable ion-dipole interactions.
Density functional theory (DFT) calculations were performed to get additional structural information on the host-guest complexation.The optimized structures for the protonated/ neutral phenylephrine as well as the non-covalent interactions (NCI) analysis are shown in Fig. 7. NCI analysis provide the visual illustration of the non-covalent interactions (Fig. 7B), such as hydrogen bonds (depicted with blue color), van der Waals interactions (depicted with green color), and repulsive   steric interactions (depicted with red color). 44The optimized structure of CB7$phenylephrine in the protonated form indicated the formation of inclusion complex, in which the phenyl ring is encapsulated inside the cavity.In addition, the complex is stabilized by ion-dipole interaction between the ammonium group and the carbonyl rim from the upper side, as well as hydrogen bonding between the hydroxyl group and the lower rim (Fig. 7A).This result is in agreement with the 1 H NMR data.For the unprotonated form of phenylephrine, the inclusion complex is stabilized by hydrogen bonding between the amine/ hydroxyl groups and the carbonyl rims (Fig. 7B).It can be seen from the NCI analysis that the protonated form of phenylephrine involved more attraction interactions compared to the neutral one.The calculated interaction energy revealed a stabilization of the protonated complex by 63 kcal mol −1 relative to the neutral one.
Phenylephrine hydrochloride extended stability at low and ready-to-use concentrations has been recognized problematic. 22,45,46Consequently, there is a need to improve stability phenylephrine hydrochloride.In this regard, supramolecular complexation provides a versatile solution for drug stability. 47he host-guest complexation between CBn and several drug molecules has shown positive impact on their stability. 16herefore, the complexation of phenylephrine hydrochloride by CB7 may serve as a potential strategy to enhance its stability and bioavailability.

Conclusions
In summary, we report on the host-guest complex between CB7 and phenylephrine in aqueous solution.CB7 forms inclusion complex with protonated phenylephrine with a high binding affinity, which is attributed to the combination of ion-dipole and hydrogen bonding interactions, as well as the hydrophobic effect.The supramolecular host-guest assembly resulted in a complexation-induced pK a shi, revealing an increase in the basicity of phenylephrine.The recognition of phenylephrine by CB7 enables the development of uorescence-based sensing assay applying the indicator displacement strategy.

Experimental details
CB7 was synthesized as previously reported. 48Phenylephrine hydrochloride and berberine chloride were purchased as a solid powder from Sigma-Aldrich (Germany).Hydrochloric acid and sodium hydroxide were used to adjust the pH.UV-Visible absorption measurements were performed with a Varian Cary 4000 spectrophotometer and the uorescence spectra were recorded on a Varian Cary Eclipse.All optical measurements were performed at ambient temperature, using a rectangular quartz cuvette with 1 cm optical path length.Proton nuclear magnetic resonance ( 1 H NMR) spectra were measured by using a 400 MHz FTNMR NanoBay spectrometer (Bruker, Switzerland) in D 2 O.
Solutions were prepared in Milli-Q water and le to equilibrate before the measurements.The binding affinity of berberine dye to CB7 was measured by using uorescence titration (pH = 3.0).The uorescence emission (l ex = 345 nm and l em = 495 nm) of berberine (20 mM, kept xed during the titration) was measured as a function of CB7 concentration and the binding constant (K affinity ) was tted according to the nonlinear curve t for a 1 : 1 binding stoichiometry. 49Indicator displacement experiment was performed to determine the binding constant of phenylephrine with CB7.In detail, phenylephrine was added to a solution of preformed CB7$berberine complex (using 35 mM of CB7 and 20 mM of berberine, both kept xed during the titration), and the uorescence emission (l ex = 345 nm and l em = 495 nm) was recorded upon each addition.The data were tted according to the non-linear curve t. 50For the pK a determination, the absorption spectra of phenylephrine (50 mM) were recorded as a function of pH in the absence and presence of 1.0 mM CB7 (to ensure a high degree of complexation). 51 1H NMR spectra for free CB7 (2.0 mM) and phenylephrine (2.0 mM), as well as the CB7$phenylephrine complex were measured in D 2 O.
Quantum-chemical calculations were performed using Gaussian 16 52 at the M06-2X/6-31+G** level of theory in the water applying the implicit solvation model based on density (smd) method. 53The non-covalent interactions (NCI) have been performed by using MULTIWFN v3.7. 44

Fig. 2
Fig. 2 Schematic representation of the fluorescent-dye displacement assay principle for analyte binding using CBn/dye reporter pair.

Fig. 3 (
Fig. 3 (A) Fluorescence emission spectra of CB7$berberine complex (l ex = 345 nm) at various concentrations of phenylephrine as a competitive guest in aqueous solution, at pH = 3.0.(B) The intensity changes (l em = 495 nm) as a function of phenylephrine concentration; the binding constant value was extracted from the fitted data as 4.0 × 10 6 M −1 .

Fig. 4
Fig. 4 Linear fluorescence response for the detection of phenylephrine in aqueous solution using CB7$berberine reporter pair.

Fig. 5
Fig. 5 UV-absorption spectra of free phenylephrine and CB7$phenylephrine inclusion complex at different pH values (A and C, respectively).Fitted pH-dependent absorbance data at 295 nm (B and D).